Atherosclerosis is considered an inflammatory disease triggered by the entry of monocytes into the wall of large arteries where they differentiate into macrophages and foam cells. Several adhesion molecules on endothelial cells, monocytes and platelets are relevant for the binding of monocytes. In order to investigate the molecular mechanisms of monocyte recruitment to atherosclerotic lesions, we have recently developed an ex vivo model in which monocytes or monocyte-like cell lines are perfused through carotid arteries isolated from apoE-/- mice, and their interaction with the vessel wall is investigated by intravital microscopy. Monocyte adhesion under flow depends on activation of integrins, which we hypothesize to be initiated by chemokines presented on the endothelial surface at lesion-prone sites. The progression of atherosclerotic disease is greatly accelerated in diabetic individuals and animal models and is associated with increased oxidative stress. The overall hypothesis of this project is that inflammatory adhesion molecules, chemokines and chemokine receptors determine monocyte recruitment to atherosclerotic lesions in mice with and without diabetes. Limiting oxidative stress may have beneficial effects on adhesion molecule and chemokine expression and function. We propose to measure the expression of adhesion molecules, chemokines, and chemokine receptors in apoE-/- mice with and without diabetes by immunostaining and flow cytometry. We will investigate the mechanism by which monocytes roll, adhere and become activated at lesion-prone sits in large arteries of apoE-/- mice by using our isolated-perfused carotid artery and response-to-injury models. We have already identified P-selectin, VCAM-1, PSGL-1 and VLA4 as key adhesion molecules and will now focus on other adhesion molecules and chemokines presented by the atherosclerotic endothelium. We will extend these studies to atherosclerotic mice with diabetes (db/db x apoE-/-) and altered oxidative stress (12-LO-/- x apoE-/-). Finally, we will test whether therapeutic interventions aimed at limiting oxidative stress will limit monocyte rolling, adhesion, and/or activation by using the novel anti-inflammatory compound lisofylline in apoE-/- mice with and without diabetes, and with and without wire injury. In addition to histology and functional studies, we will add magnetic resonance imaging to serially monitor lumen size of mouse carotid arteries. This technique was recently established in our laboratory. These studies are designed to provide mechanistic insight into monocyte recruitment to atherosclerotic lesions and the mechanisms by which diabetes accelerates atherosclerosis.